StarD7 deficiency hinders cell motility through p-ERK1/2/Cx43 reduction

StarD7 belongs to START protein family involved in lipid traffic, metabolism, and signaling events. Its precursor, StarD7.I which is important for mitochondrial homeostasis, is processed to the StarD7.II isoform that lacks the mitochondrial targeting sequence and is mainly released to the cytosol. StarD7 knockdown interferes with cell migration by an unknown mechanism. Here, we demonstrate that StarD7 silencing decreased connexin 43 (Cx43), integrin β1, and p-ERK1/2 expression in the non-tumoral migratory HTR-8/SVneo cells. StarD7-deficient cells exhibited Golgi disruption and reduced competence to reorient the microtubule-organizing center. The migratory capacity of StarD7-silenced cells was reestablished when Cx43 level was resettled, while p-ERK1/2 expression remained low. Importantly, ectopic expression of the StarD7.II isoform not only restored cell migration but also ERK1/2, Cx43, and integrin β1 expression. Thus, StarD7 is implicated in cell migration through an ERK1/2/Cx43 dependent mechanism but independent of the StarD7.I function in the mitochondria.


Introduction
Cell migration is crucial in a variety of physiological processes comprising embryonic development, immune surveillance, wound healing, and trophoblast invasion. Dysregulated migration is detected in many pathologic conditions such as inflammation, cancer, and also in pregnancy complications including pre-eclampsia, intrauterine growth restriction, placenta accreta spectrum, and gestational trophoblastic disease. Cell migration is a tightly regulated, multi-step, and highly dynamic process that involves a growing number of signaling pathways, cell surface receptors, transcription factors, cytoskeleton proteins, integrins, extracellular matrix (ECM), and ECM-associated proteins [1][2][3][4].
Connexin 43 (Cx43) is a member of a large family of integral membrane proteins that establish Gap Junction Intercellular Communication (GJIC) permitting the intercellular transference of ions, metabolites, and small signaling molecules [5]. Also, Cx43 forms hemichannels allowing the passage of molecules up to 1 kDa from the cytoplasm to the extracellular milieu and vice versa [6]. Besides the GJIC function, connexins have channel-independent functions affecting cell morphology through the cytoskeleton rearrangement controlling cell polarity and cell migration [2,7,8]. StarD7, a member of START lipid transfer protein family, was initially described in the trophoblast-derived JEG-3 cell line [9]. StarD7 is synthesized as a precursor protein named StarD7.I that contains a mitochondrial localization signal which is rapidly cleaved originating a shorter protein named StarD7.II mainly released to the cytosol [10,11]. StarD7.I delivers phosphatidylcholine to the mitochondria [11][12][13][14]; its contribution to the trophoblast physiology [15] as well as in preserving endoplasmic reticulum (ER) and mitochondrial morphology and dynamics were established [11,13,14,16,17]. At present, a specific role of the abundant cytosolic StarD7.II isoform has not been reported. We have previously demonstrated that StarD7 knockdown reduces cell migration and Cx43 expression in JEG-3 cells [15,18]; however the mechanism involved remains unexplored. Here, we used the migratory and nontumoral HTR-8/SVneo cell line to uncover the mechanism and downstream targets. The study highlighted the role of the StarD7.II isoform in regulating polarized cell migration.
The results show that StarD7-dependent reduction in cell migration relates to ERK1/2/ Cx43 expression. Furthermore, our findings indicate that StarD7 silencing causes a disruption of the Golgi apparatus and a deficiency in cell polarity. Through StarD7.I, StarD7.II, and Cx43 recovery assays, we demonstrate that the reduced cell migration in StarD7-deficient cells is dependent on Cx43 expression but independent of the well-known function of StarD7.I in the mitochondria. Altogether these results reveal a novel role for StarD7.II isoform in regulating cell migration.
Alternatively, HTR-8/SVneo cells were stably transduced with a short hairpin RNA (shRNA) vector that targets the sequence CGGTTGGAAGAAATGTCAAAT located at position 711-731 of the StarD7 transcript. To this end, lentiviral particles were generated by transfecting HEK-293T cells with psPAX2, pMD2.G (Addgene) and pLKO.1 containing the shRNA sequence (shD7) against StarD7 (The RNAi Consortium clone ID TRCN0000151458, Sigma-Aldrich) or pLKO.1 empty (shC) vectors (SHC001, Sigma-Aldrich). Conditioned medium with viruses was collected after 72 h and used to transduce HTR-8/SVneo cells as described [13]. Cells were collected and StarD7 levels analyzed by Western blot and qRT-PCR. Stable cells were amplified and used for subsequent experiments with less than 5 passages.

Cell migration assay
HTR8/SVneo cell migration was determined by wound healing assays and performed in siC and siD7 as previously described [15]. To perform cell migration assay in stable shC and shD7 cells, they were seeded on twelve-well plates (1 × 10 5 cells/well) and cultured to reach a confluence of 70%. Then, cells were transfected or not with EV-GFP, p-Cx43-GFP, EV, D7.I, or D7. II plasmids for 48 h, as indicated. Confluent monolayers were wounded and assessed under microscopy at 0 and 8 h after. The results were expressed as the percentage of the wound closure calculated as the difference between the remaining area at 8 h and the initial one at 0 h recorded in seven fields of duplicate wells from three independent experiments.
Analysis of the migration of individual cells was conducted employing an IN Cell Analyzer 2500 HS (High content analysis, GE Healthcare Life Sciences) in a time-lapse mode (one picture every 15 min interval over a total time of 120 min). Individual cell trajectories were manually tracked using Image J/ Fiji software. Only cells at the front line of migration were examined, using the border of the cell as the reference mark for cell movement. Movies are presented in the S1-S3 Movies.

RNA extraction and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was purified with Trizol (Thermofisher), following the manufacturer's instructions. Single-stranded cDNAs were synthesized and qRT-PCR was carried out with SYBR Green PCR Master Mix (Applied Biosystems) using the primers for StarD7 and cyclophilin A as previously described [13]. The transcript levels were normalized to those of cyclophilin A and the relative expression levels were quantified as described [13]. Amplification efficiency for each set of primers was near 98%. No amplification was detected in PCR reactions using water instead of template.
For microtubule-organizing center (MTOC) orientation analysis, a wound was conducted on confluent StarD7-silenced or control cell monolayers and 6 h later the cells were fixed and stained for γ-tubulin (1:2000) and Hoechst 33342 as described above. Cells with MTOCs located in the 120-degree sector facing the leading edge were considered oriented and cells with MTOCs positioned outside this sector were recorded as not oriented as described [21]. Cells located at the border of the wound were examined, and percentages were obtained from 350 cells per condition and assay.
Cx43 mean fluorescent intensity (MFI) was quantified using Image J/Fiji software over 50 cells per condition from images obtained at 630x in Fluorescence microscope DMI 8 (LEICA, Wetzlar, Germany).

Data analysis
All quantitative data were expressed as mean ± SEM of independent experiments and analyzed using the one sample t-test, Student's t-test, or one-way ANOVA as indicated. Dunn's posttest was performed for multiple comparisons of independent samples. Significance was taken as p< 0.05.

StarD7 deficiency leads to reduced expression of Cx43 and other proteins involved in cell migration
We have previously documented that StarD7 silencing with a siRNA inhibits cell migration in JEG-3 cells [15]. Herein, we studied the underlying mechanism using the non-tumoral HTR-8/SVneo cell line characterized by a migratory phenotype. StarD7 siRNA (siD7) and shRNA (shD7) were used to deplete StarD7 levels. We verified that in both experimental conditions StarD7 protein levels were significantly decreased by > 90% and 60%, respectively ( Fig 1A). Also, reduced StarD7 mRNA expression was determined by qRT-PCR ( Fig 1B). As reported in JEG-3 cells, transient transfection with siD7 reduced HTR-8/SVneo cell migration evaluated by wound healing assays. In addition, cell migration was also reduced in stable StarD7-silenced HTR-8/SVneo cells ( Fig 1C).
As mentioned, earlier exploratory data suggest that Cx43 mRNA is largely downregulated in JEG-3 StarD7-silenced cells [18]. In addition, numerous reports have linked Cx43 down- regulation with reduced cell motility and invasion [2,7,8]. Therefore, we explored Cx43 expression in HTR8/SVneo cells where StarD7 expression was decreased by siRNA or shRNA. We found that StarD7 silencing led to a reduction in Cx43 protein levels in both experimental conditions (Fig 2A). Three bands were detected on Western blots indicating phosphorylation of Cx43, as reported [22,23]. Accordingly, abundant punctuate Cx43 immunostaining was observed in control samples while a significant MFI reduction was noticed in StarD7-silenced cells, confirming the data obtained from Western blot analysis (Fig 2B). Considering that Cx43 interacts with ECM and ECM-associated proteins connected to the migration process [7,[24][25][26][27], we assessed whether the reduced cell migration induced by the loss of StarD7 also affects the expression of well-known key proteins connected with this process such as integrin β1, integrin α5, and p-FAK. Western blot assays revealed that the amount of all the assayed proteins was reduced in shD7 cells compared to shC cells (Fig 2C).

The decrease in HTR-8/SVneo cell migration was accompanied with a reduction in the ERK pathway activation
Numerous evidences reveal that ERK pathways are required to promote cell motility [28,29]. In addition, several reports indicate that Cx43 is involved in regulating ERK1/2 signaling [30][31][32][33][34]. Thus, we explored the expression of p-ERK1/2 in StarD7-silenced cells. As illustrated in

PLOS ONE
StarD7-deficiency reduces ERK1/2/Cx43 signaling  Fig  3A). Interestingly, when shD7 cells were treated with 20 or 40 ng/mL of EGF during 8 h, the amount of Cx43 was increased in comparison with non-treated cells, indicating that the ERK1/2 pathway is upstream of Cx43 expression (Fig 3B). Additionally, p-ERK1/2 levels also increased following the treatment with 15 ng/mL of EGF in shD7 as in shC cells indicating that this signaling pathway is conserved in shD7 cells (Fig 3C).

StarD7 silencing causes a deficiency in cell polarity and a disruption of the Golgi apparatus
Considering that Cx43 deficiency causes a failure of the MTOC to reorient with the direction of wound closure [21,35], we investigated the location of MTOC during polarized cell migration. To this end, wound healing assays were carried out in StarD7-silenced and control cells and the MTOC was visualized through γ-tubulin immunofluorescence staining. In control cells the MTOC was principally located towards the direction of cell migration, as expected. In contrast, in the shD7 and siD7 conditions, the percentage of cells with oriented MTOC was reduced indicating a deficiency in cell polarity (Fig 4A and S1A Fig).
Taking into account that the directional cellular locomotion depends on the integrity of the Golgi apparatus [36], we explored Golgi morphology in StarD7-silenced cells by immunofluorescence staining of GM130, a cis-Golgi resident protein. As expected, GM130 staining was localized in the Golgi complex showing a condensed and perinuclear pattern in control cells (siC or shC) whereas in StarD7-silenced cells, GM130 signal appeared as a punctuated dispersed pattern indicative of Golgi disruption (Fig 4B and S1B Fig). Quantification showed that 49.0 ± 3.0% of shD7 cells exhibited a disrupted Golgi phenotype compared to only 27.0 ± 3.0%

Exogenous Cx43 restores cell migration but not ERK1/2 phosphorylation in StarD7-deficient cells
To confirm that the altered phenotype observed in StarD7-deficient cells was due to the Cx43 reduction, we restored Cx43 levels by exogenous addition. To this end, we transfected shD7 cells with the pCx43-GFP or EV-GFP plasmids and analyzed the capacity of cells to migrate in

PLOS ONE
StarD7-deficiency reduces ERK1/2/Cx43 signaling migration recovery of StarD7-deficient cells when Cx43 levels were increased, further highlighting that StarD7 modulates cell migration through Cx43 (Fig 5B and S1-S3 Movies). Interestingly, the p-ERK1/2 levels in shD7 cells were not modified by the addition of exogenous Cx43 compared to shD7 cells transfected with EV-GFP (Fig 5C), further supporting that the ERK1/2 pathway is upstream of Cx43 expression since, as mentioned above, ERK1/2 activation was able to increase Cx43 expression in shD7 cells (Fig 3B). Collectively these data suggest that StarD7 regulates polarized cell migration through Cx43 and suggest a mechanism that involves the p-ERK1/2/Cx43 signaling pathway.

Exogenous StarD7.I and StarD7.II expression restores Cx43, integrin β1, p-ERK expression and cell migration
In order to confirm that the reduced expression of Cx43 and of the other reported proteins, as well as cell migration impairment is a consequence of StarD7 deficiency, we performed rescue experiments. To this end, StarD7 knockdown cells were transfected with plasmids encoding the StarD7.I or StarD7.II isoform (D7.I and D7.II, respectively) or with the EV control. Transfection with either D7.I or D7.II plasmids restored mature StarD7 expression to a level like that observed in non-silenced shC cells, as demonstrated by western blot (Fig 6A). The detection of the mature StarD7 isoform after transfection with the D7.I plasmid was as expected, since the StarD7.I protein has an N-terminal mitochondrial localization signal which is rapidly processed in the mitochondria originating the mature StarD7.II isoform [11,13,14,16,17]. Exogenous expression of StarD7.I reestablished Cx43, integrin β1, and p-ERK1/2 protein to levels comparable to those observed in shC cells, while the expression of these proteins remained unmodified in cells transfected with the EV control ruling out a nonspecific effect due to the vector or the transfection protocol (Fig 6A and 6B). Remarkably, the exogenous expression of the StarD7.II isoform also restored protein levels to those observed in shC cells (Fig 6A and 6B). These results confirm that reduced Cx43, integrin β1, and p-ERK1/2 protein levels are due to StarD7 silencing. Moreover, cell migration was also recovered in StarD7-silenced cells transfected with either isoform, further confirming that the altered phenotypes observed in knockdown cells are a consequence of StarD7 deficiency (Fig 6C). Interestingly, since exogenous expression of StarD7.II restores cell phenotype, and this isoform is lacking the mitochondrial targeting sequence [11,13,14,16,17], present results suggest StarD7 role in cell migration is independent of the function of StarD7.I in the mitochondria.
To rule out that the reduction in cell migration is dependent on the well-documented role of StarD7.I in supporting mitochondrial activity [11,13,14,16,17], we measured mitochondrial ROS levels and the Δψm, as indicator of mitochondrial activity. Δψm and mitochondrial ROS levels were not modified in shD7 cells as compared to the control (S2 Fig).

Discussion
This study provides an understanding of the role of StarD7 in HTR-8/SVneo cell migration and uncovers the main targets mediating StarD7 effects on its motility behavior. Herein, through siRNA/shRNA silencing, and protein rescue experiments, we show that StarD7 deficiency leads to a reduction in Cx43 expression resulting in defects in polarized cell migration.
Cell migration is a complex, multi-step, and highly dynamic process that involves a growing number of signaling pathways, cell surface receptors, transcription factors, cytoskeleton proteins, integrins, ECM, and ECM-associated proteins, in addition to Gap junction proteins [1][2][3][4]. Interestingly, a number of evidences clearly indicate that Cx43 interacts with ECM and ECM-associated proteins connected to the migration process [7,[24][25][26][27]. Present results show that StarD7-dependent Cx43 downregulation was associated with a decreased integrin α5, integrin β1, and p-FAK protein levels. In addition, StarD7 knockdown decreased ERK1/2 phosphorylation. Numerous evidences reveal that ERK pathways play an important role in promoting cell motility [28,29]. Although, several studies indicate that ERK1/2 signaling pathway regulates Cx43 [37][38][39][40][41]; other reports involves Cx43 in the regulation of ERK1/2 signaling pathway [30][31][32][33]. Our data demonstrate that ERK1/2 phosphorylation was recovered by the addition of either StarD7 isoforms but not by exogenous Cx43, indicating that in this cell model ERK1/2 phosphorylation is upstream of Cx43. Remarkably, the addition of EGF to shD7 cells not only increased ERK1/2 phosphorylation, but also Cx43 levels reinforcing the idea that StarD7 silencing reduces Cx43 expression via ERK1/2 signaling pathway resulting in reduced cell motility. In this regard, Hino et al. have demonstrated the importance of the periodic ERK activation in the form of waves for collective cell migration [42]. Although it was not evaluated, it is possible that increased Cx43-mediated cell communication contributes with ERK waves transferring signaling molecules that enhance polarized cell migration. Alternatively, ERK-induced Cx43 expression might enhance cell migration through its channel-independent function known to control cytoskeleton morphology, directional migration, and organelle polarity, acting as a signaling molecule [2,7,8,21,43]. In this sense, Zhao et al. reported that betacellulin, a member of the EGF family, induces Cx43 expression through the activation of the MEK-ERK signaling pathway promoting ovarian cancer cell migration, and this effect is likely gap junction-independent [37]. In addition, we cannot discard the participation of Cx43 or other membrane channel proteins, such as Pannexins, that contribute to ERK signaling during migration through the release or uptake of ATP [44,45].
It is well-known that directional cell migration is determined by the polarized distribution of protrusions at the cell periphery; and that cell polarity, which is a precondition for directed cell migration, is altered after Golgi apparatus disruption by pharmacological inhibition or by knocking-down structural proteins [46,47]. Here, we show that StarD7 suppression leads to decreased Cx43 protein levels, Golgi apparatus disruption and a reduced cell capability to reorient the MTOC in a polarized migration. Our results are in line with the defects in cell polarity observed in mouse embryonic fibroblasts [21] and mouse epicardial cells [35] where a decrease in Cx43 levels, breakdown in the localization of the Golgi apparatus and disoriented MTOC next to the wound closure were detected. Importantly, the exogenous expression of Cx43 in StarD7-depleted cells was able to restore the motility cell behavior indicating that reduced StarD7 levels impair polarized cell migration, at least in part, through Cx43.
Furthermore, re-establishing the amount of StarD7.I or StarD7.II protein substantially restored Cx43, integrin β1 and p-ERK1/2 levels to those observed in the non-silenced cells accompanied by a recovery of cell migration, confirming the specificity of the effect. In addition, our data reveal that the migratory phenotype observed in StarD7-deficient cells is not associated with an important mitochondrial bioenergetic defect since mitochondrial membrane potential as well as mitochondrial ROS levels were not altered. More significantly, they indicate that the reduction in cell migration, which is modulated by Cx43 expression level, is due to a decreased StarD7.II isoform level, and is independent on the role of StarD7.I in mitochondrial morphology and dynamics [11,13,14,16,17]. Although we cannot rule out that StarD7 deficiency engages cell migration by inflicting other forms of mitochondrial damage, our data favor a model in which StarD7.II isoform deficiency followed by Cx43 reduction is responsible for the cell migration defects observed.
It has been reported that StarD7 haploinsufficient mice present alterations in lung epithelial barrier function with an enhanced allergic response, spontaneous atopic dermatitis, and decreased amount of the key tight junction protein zonula occludens-1 (ZO-1) [14,48]. Interestingly, Erythrokeratodermia Variabilis et Progressiva [49] and severe dermatitis, multiple allergies, and metabolic wasting syndrome were reported to be caused by mis-localized Cx43 or enhanced Cx43 turnover [50]. Considering that ZO-1 is a well-known protein able to interact with Cx43 [51], it is attractive to hypothesize that the alteration in the epithelial barrier functionality observed by Yang et al. [48] in StarD7-knockdown airway cells could be a consequence of Cx43 reduction. Moreover, Yang et al. reported that the majority of StarD7 −/− mice died between embryonic day E10 and E11 most likely due to the disruption of cardiovascular development [48]. Similarly, Cx43 knockout mice die soon after birth as a consequence of cardiac defects connected with pulmonary outflow obstruction and coronary abnormalities [35,52,53]. Therefore, we speculate that the cardiac defects observed in StarD7 −/− mice could be due, at least in part, to a decreased Cx43 expression. Furthermore, several reports clearly indicate that the development and function of the female reproductive tissues are regulated by connexins [54,55]. Indeed, recurrent early pregnancy loss risk was linked with a decreased Cx43 transcript and protein expression in trophoblast cells [56,57], highlighting the necessity to explore the StarD7 role in placental disorders.
Collectively, these findings reveal that StarD7 depletion causes a reduction in p-ERK1/2/ Cx43 levels impacting on cell migration behavior as well as in the expression of other proteins involved in this process, revealing a novel role for StarD7.II isoform (Fig 7).
Supporting information S1 Fig. siRNA-mediated StarD7 silencing impairs MTOC reorientation and disrupts the Golgi apparatus. (A) The monolayer of siC or siD7 cells was wounded and 6 hours later cells were stained with anti-γ-tubulin (red) to detect MTOC (arrows). The boxed regions are enlarged in the right panels. The nuclei were labeled with Hoechst (blue), merged images are shown on the left. The images were recorded by fluorescence microscopy and white lines indicate the wound edge. Scale bar = 10 μm (600x). Bar graph shows the percentage of siC and siD7 cells exhibiting oriented MTOC after wound from two independent experiments (mean ± SEM, � p< 0.05, Student's t-test). (B, C) Golgi apparatus morphology was visualized by fluorescence microscopy in siC and siD7 cells using anti-GM130 or anti-Golgin 97 antibodies, respectively (red). The nuclei were labeled with Hoechst (blue). Scale bar = 30 μm (200x). The boxed regions are enlarged in the right panels. Bar graph shows the percentage of siC and siD7 cells with fragmented Golgi from two independent experiments (mean ± SEM, � p< 0.05, Student's t-test). (TIF)